Anti-phosphatidylethanolamine (aPE) is an autoimmune condition characterized by the presence of circulating antibodies against phosphatidylethanolamine (PE), and is associated with clinical symptoms of thrombosis and repeated pregnancy loss. Currently, the pathogenic mechanism for aPE is unknown. PE is an abundant phospholipid in cellular membranes. However, since PE is sequestered inside the cell, its absence from the cell surface raises the question how PE is accessible as an antigenic target to circulating anti-PE antibodies. For the first time, the current investigations discovered that PE-binding agents target the inner membrane of early endosomes, which indicates that cells may be vulnerable to endocytosed anti-PE antibodies. These findings were obtained using novel PE-specific molecular probes, binding agents and purified anti-PE antibodies from aPE patients. Based on the preliminary data, we propose the central hypothesis that the endosomes are the primary target to anti-PE antibodies for the pathogenic stimulation of inflammatory signaling. We will test the hypothesis in two Specific Aims. In Aim 1 we will examine the impairment of endosomes in response to PE- binding agents, and investigate the roles of type IV P-type ATPases in PE translocation in endosomes. In Aim 2, we will identify the signaling pathway that leads to reactive oxygen species (ROS)-mediated and TNF-?- dependent upregulation of pro-inflammatory factors. The research plan will focus on a novel mechanism of endosomal NADPH oxidase activation as a consequence of PE binding/sequestration. Overall, the outcome of this investigation will shed light on the pathogenesis of anti-PE antibodies, which ultimately will have a positive impact on the clinical diagnosis and treatment of aPE.